Composition and method for inner ear sensory hair cell regeneration and replacement

ABSTRACT

A composition and method for replacement and regeneration of hair cells of the inner ear is provided. The composition comprises an active agent in an amount effective to decrease Hes1 gene expression in a tissue of the inner ear. The active agent can be short interfering RNA (siRNA) molecules encapsulated in a biodegradable nanoparticle. The method involves administering a solution to the inner ear where the solution contains an active agent in an amount effective to decrease Hes1 gene expression.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/701,550 (National Stage of PCT/US2011/038926), filed Feb. 18, 2013,incorporated herein by reference in its entirety, which claims priorityfrom Provisional application U.S. Application 61/351,623, filed Jun. 4,2010, incorporated herein by reference in its entirety.

BACKGROUND

Deafness and balance dysfunction are common human disabilities. In themajority of cases these disabilities result from the loss of sensoryhair cells in the (1) organ of Corti (OC) in the cochlea, (2) thevestibular epithelium in the cristae or (3) saccule or utricle of thevestibular organ. Currently there is no FDA approved treatment that cancure these disorders by restoring the sensory hair cells in thesetissues.

Current approaches to the problem involve vestibular rehabilitation toallow adaptation to the injury to the vestibular organs. Therehabilitation is time consuming, and does not restore lost function.For sensorineural deafness, rehabilitation can be achieved with hearingaids or cochlear implants. However, these devices are expensive, requirean extensive surgery and produce a subnormal sound quality and onlypartial return of function.

Another approach in treating hearing disorders is administration ofpeptides or other small molecules. Often treatment results are limitedwith the use of such due to relatively high cochlear concentrations thatmust be achieved (micro or millimolar). Moreover, protein or peptideinhibitors are difficult to deliver systemically to treat the ear due tothe blood labyrinthine barrier and protein clearance in the bloodstreamas well as potential antigenicity. Difficulties also exist in terms ofdelivering adequate concentrations of peptide and protein directly tothe cochlea as well, particularly using topical delivery due to the sizeof the molecule.

One potential alternative to these traditional approaches is usingtargeted gene therapy to induce inner ear hair cell regeneration andreplacement. For example, hair cell regeneration or replacement has beenachieved in rodents through the use of a viral vector to introduce theAtoh1 gene into inner ear sensory epithelium. However, this approachcarries risk inherent in viral vector therapy including the induction ofinfection, an inflammatory immune response, genetic mutation,development of neoplasia and others. Silencing of kip1p27 RNA has beenshown to induce hair cell regeneration but in an ectopic fashion withoutreturn of function. Modulation of the retinoblastoma gene can alsoproduce additional hair cells but there may be danger inherent inmanipulating an oncogene, or cancer causing gene. Thus, current genetherapies directed to regeneration or replacement of inner ear haircells have failed to identify a safe and effective molecular target anddelivery method.

One potential gene therapy approach is through the use of shortinterfering RNA (siRNA). Once introduced into a cell, the siRNAmolecules complex with the complimentary sequences on the messenger RNA(mRNA) expressed by a target gene. The formation of this siRNA/mRNAcomplex results in degradation of the mRNA through a naturalintracellular processes known as RNA interference (RNAi). RNAi is awell-established tool for identifying a gene's function in a particularcellular process and for identifying potential therapeutic targets indisease models. Although RNAi has traditionally been used in cellculture and in vitro applications, gene-therapy based therapeutics arenow being explored utilizing this process.

As discussed above, several gene targets have been explored with respectto regeneration of hair cells of the inner ear without much success. Thebasic helix-loop-helix (bHLH) genes Hes1 and Hes5 have been identifiedas playing roles in sensory hair cell development in the cochlea andvestibular structures of the ear. In addition, a potential gene targetfor preventing loss of hair cells is mitogen-activated protein kinase 1(MAPK1), which plays a role in programmed cell death or apoptosis.However, the potential for these to be effective therapeutic targets forregeneration or protection of sensory hair cells of the inner ear hasyet to be demonstrated and or identified as a viable approach.

SUMMARY

To address the deficiencies in the current treatment options for hearingand other inner ear-related disorders, the compositions and methodsdescribed herein provide a safe and effective means to promote thereplacement, regeneration, or protection of sensory hair cells of theinner ear.

In one embodiment, a composition to regenerate hair cells of the innerear is provided. The composition comprises an agent to decrease targetgene expression encapsulated or incorporated into a nanoparticle. Theagent is in an effective amount to decrease the expression of targetgenes selected from the group consisting of Hes1, Hes5, and MAPK1. Thepreferred nanoparticle comprises a biocompatible and biodegradablepolymer and is more preferably poly(lactic-co-glycolic acid) (PLGA). Inone aspect, the agent comprises one or more siRNA molecules sufficientto decrease the mRNA levels of Hes1, Hes5, or MAPK1.

In another embodiment, the nanoparticle further comprisessuperparamagnetic iron oxide nanoparticles (SPION) coated with oleicacid in order to render the nanoparticle susceptible to movement ortransport by applied magnetic gradients to a desired location of theinner ear. Furthermore, nanoparticles comprising SPION can be used toconfirm proper localization of the nanoparticle in the target tissueusing, for example, magnetic resonance imaging (MRI).

In a separate embodiment, a method for regenerating sensory hair cellsof the inner ear is provided. The method comprises the steps of: a)applying a solution directly to the inner ear, wherein said solutioncomprises a suspension of nanoparticles, wherein the nanoparticles haveincorporated therein an agent to decrease the expression of the genesselected from the group consisting of Hes1, Hes5, and MAPK1.

In another embodiment, the solution is applied to the middle ear. Inthis embodiment, the nanoparticles comprise SPION and one or more siRNAmolecules in an amount effective to decrease the expression of Hes1,Hes5 or MAPK1 and the method further comprises the step of applying amagnetic force to enhance the transport of the magnetic nanoparticlesacross the round window membrane and into the inner ear.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides transmission electron microscopy images of explantedguinea pig utricular maculae from the following treatment groups: (1)control (scrambled control siRNA)—images A, E; (2) neomycin—images B, F;and (3) neomycin+Hes1 siRNA—images C, D, G, H.

FIG. 2 is a bar graph representing the mean±SEM pDNA (pg) isolated fromthe cochlea of guinea pigs administered a solution containing PLGAnanoparticles carrying a pDNA and SPION payload in the presence andabsence of an external magnetic force.

FIG. 3 is a bar graph representing the mean t: SEM number of immaturehair cells (new hair cells) present in explanted guinea pig utriclesfollowing toxin-induced injury (Neo or 4-HNE) in the presence andabsence of transfected Hes1 siRNA.

FIG. 4 provides 7 Tesla MRI images of cochlea from guinea pigs in thefollowing treatment groups: (1) control—image A; (2) solution containingPLGA nanoparticles carrying a SPION payload administered to the roundwindow membrane—image B; and (3) solution containing PLGA nanoparticlescarrying a SPION payload administered to the round window membrane inthe presence of an external magnetic field.

FIG. 5 provides 7 Tesla MRI images of cochlea from guinea pigs in thefollowing treatment groups: (1) control—image A; (2) solution containingPLGA nanoparticles carrying a SPION payload administered to the roundwindow membrane—image B; and (3) solution containing PLGA nanoparticlescarrying a SPION payload administered to the round window membrane inthe presence of an external magnetic field.

FIG. 6 is a bar graph representing Hes1 mRNA levels as a percentage ofcontrol from P3 CD-1 mouse cochlea transfected with either scrambledcontrol siRNA (control) or Hes1 siRNA.

FIG. 7 is a bar graph representing the mean±SEM number of hair cellspresent in cultured P3 CD-1 mouse Organ of Corti following toxin-inducedinjury (4-HNE) in the presence and absence of transfected Hes1 siRNA.

FIG. 8 is a bar graph representing the mean±SEM number of hair cellspresent in cultured P3 CD-1 mouse Organ of Corti following toxin-inducedinjury (4-HNE) in the presence of varying concentrations of PLGAnanoparticles carrying a Hes1 siRNA payload.

FIG. 9 is a bar graph representing the mean±SEM number of hair cellspresent in cultured P3 CD-1 mouse saccules following toxin-inducedinjury (neomycin) in the presence of varying concentrations of either(1) PLGA nanoparticles carrying a MAPK1 siRNA payload or (2) transfectedMAPK1 siRNA.

DETAILED DESCRIPTION

As used herein, “inner ear” includes, but is not limited to thefollowing structures: auditory labyrinth; vestibular labyrinth includingthe vestibular ganglion, cochlear ducts, and endolymphatic sac; cochleartissues including Organ of Corti, spiral ganglion, and spiral ligament;tissues of the endolymphatic duct; tissues of the stria vascularis;utricle tissues including urticular maculae and saccular maculae; andepithelial tissue of the cristae ampularis.

As used herein, the terms “a” and “an” mean “one or more”.

As used herein, the term “active agent” means a therapeutic agent,including but not limited to chemotherapeutic agents, radiotherapuetics,gene therapeutic agents such as siRNA molecules or other nucleic acids,an agent to interact with an intracellular or surface protein, a proteinor peptide chain, a peptide or steroid hormone, a soluble or insolubleligand, or a carbohydrate.

As used herein, the term “gene” means a unit of DNA that encodes a geneproduct such as a mRNA, functional protein, polypeptide or peptide.Thus, the term “gene expression” means the production of a gene product.For example, siRNA modifies gene expression by decreasing the amount ofmRNA available for production of a protein.

As used herein, the term “pool of siRNA molecules” means two or moredifferent siRNA molecules (directed to different subsequences on thetarget mRNA) combined together in a common payload or sample. Moreover,it should understood that a pool will consist of multiple copies of eachof the different siRNA molecules (i.e., 100 copies of siRNA molecule 1and 100 copies of siRNA molecule 2 constitutes a pool of siRNAmolecules).

The embodiments of the current invention are directed to compositionsand methods of replacement, regeneration or protection of hair cells ofthe inner ear. In a preferred embodiment, a composition for regenerationof hair cells comprises a biodegradeable nanoparticle containing asufficient amount of siRNA to decrease mRNA levels associated with theHes1, Hes5 or MAPK1 gene. Hes1 and Hes5 have been shown to play crucialroles in the regulation of sensory hair cell proliferation anddifferentiation as demonstrated by Zine et al., J Neurosci. July 1;21(13):4712-20 (2001) which is incorporated herein by reference.However, the potential for Hes1 and/or Hes5 as a therapeutic target forthe regeneration of sensory hair cells is not clear based on somewhatconflicting studies by Batts et al., Hear Res 249(1-2): 15-22 (2009) andHartman et al., JARO 10: 321 340 (2009), both of which are incorporatedby reference herein.

In a related embodiment, a composition for protection of hair cells isprovided. The composition comprises a biodegradeable nanoparticlecontaining siRNA molecules directed to various genes involved in celldeath or apoptosis. A variety of cell death pathways are activated afterinjury to the cochlea as well as inner ear balance organs which includecochlear aging processes and presbystasis. Inhibition of these celldeath processes can prevent a degree of the injury thereby increasingthe effectiveness of the regenerative treatment strategies described Inother words, treatment strategies that can stop or prevent ongoing orfuture activation of these cell death processes will enhanceregenerative treatments or serve as a useful treatment when appliedprior to or concurrently with regenerative strategies.

Some examples of cell death genes that could be targeted usingembodiments of the current invention include: caspase-mediated celldeath pathways/proteins; the tumor necrosis factor (TNF) family ofproteins; the JNK signaling pathway (mitogen activated protein kinase 1(MAPK1)/c-Jun-N-terminal kinase (JNK) cell death signal cascade);protein/peptide mediators of necrosis-like programmed cell death;proteins associated with the Poly(ADP-ribose)polymerase-1 (PARP-1)pathway required for apoptosis-inducing factor (AIF) release frommitochondria in caspase-independent apoptosis; trophic factors such asGDNF, FGF, BDNF; proteins that arrest the cell death processes caused byinner ear injury, such as cell death inhibiting peptide AM-111; andproteins and peptides associated with the Bak-dependent mitochondrialapoptosis program. Other potential targets include Bax, Bcl-x1, Bcl-2and TNFR1, calpain I and calpain II, and active cathepsin D. Using theembodiments disclosed herein, multiple cell death pathways can beinhibited by use of multiple siRNAs in a single nanoparticle payload oralternatively, by utilizing two or more nanoparticles each having adifferent siRNA payload. Moreover, the nanoparticle payloads of thecurrent invention can include siRNAs directed to genes involved in celldeath pathways in combination with siRNAs directed to Hes1 or Hes5.

In a preferred aspect of this embodiment, a biodegradeable nanoparticleis loaded with siRNA sufficient to decrease the expression of MAPK1.Cochlear injury can result in programmed cell death (apoptosis). Theinjury can result from mechanical trauma, blast trauma, acoustic trauma,infection, inflammation, toxins, chemotherapy agents such as cisplatin,certain antibiotics such as those included in the aminoglycoside family,and the aging process. The JNK/MAPK signaling pathway is involved withprogrammed cell death in most of these pathologic processes. Therefore,inhibition of cell death processes associated with JNK/MAPK activationconstitute a potential therapeutic armamentarium.

In a preferred embodiment, the active agent used to decrease target geneexpression is siRNA. The siRNA molecules directed to Hes1, Hes5 andMAPK1 mRNA, for example, can be obtained from any number of sourcesincluding Santa Cruz Biotechnology (Santa Cruz, Calif.). The siRNAmolecules described herein are generally 19-25 nucleotide-long doublestranded RNA molecules with a two nucleotide overhang on the 3′ end ofeach strand. It should be noted that these siRNA molecules can beendogenously produced in a cell through transfection of plasmid DNAsthat encode for a precursor (short hairpin RNAs) to the desired siRNAmolecules or by other various methods known by one of skill in the art.The preferred siRNA molecules directed to Hes1, Hes5, and MAPK1 aredescribed in more detail in the Examples provided below.

In a preferred embodiment, the siRNA molecules are incorporated into ananoparticle (10-300 nm based on SEM measurements) of a biocompatibleand biodegradable polymer such as PLGA using the method described byWoodrow et al. Nature Materials. 8(6):526-33 (2009), which isincorporated herein by reference. The method described by Woodrow et al.permits the loading of several hundred to 1000 molecules of siRNA pernanoparticle (several micrograms per milligram of polymer) which hasbeen shown to effectively silence target gene expression in vivo.However, the invention should not be limited to PLGA and anybiocompatible and biodegradable polymer known to one of skill in the artcould be used so long as it can encapsulate and sufficiently deliver thegene expression regulating agent to the target tissue without rejection.In one embodiment, the nanoparticles are loaded with pools of two ormore siRNA molecules, each specifically targeted to a differentnucleotide subsequence of the target mRNA molecule.

Briefly, the method described in Woodrow et al. involves a doubleemulsion solvent evaporation technique. The siRNA molecules arestabilized using natural polyamines such as spermidine (Spe). Thecomplex formation between siRNA and Spe is carried out at roomtemperature for 15 minutes on a rotary shaker. The siRNA (25-200 nmoles)is combined with Spe at a molar ratio of the Spe nitrogen to thepolynucleotide phosphate (N/P ratio) of 3:1, 8:1, and 15:1. Two hundredmicroliters of stabilized siRNA (therapeutic payload) solution inTris-EDTA buffer is emulsified into 2 mL of PLGA (100 mg)/chloroformsolution for 60 seconds on ice using a probe sonicator to form a primarywater-in-oil emulsion. This primary emulsion is re-emulsified by adding6 mL of 2% polyvinyl alcohol (PVA). The system is sonicated again for 5minutes and stirred for approximately 3-6 hours to allow chloroform toevaporate. The resultant nanoparticle solution is centrifuged at 15,000rpm for 30 minutes at 4 OC. The particles are washed with nanopure waterto remove any excess of PVA. The resultant nanoparticle pellet isdispersed in a desired volume of nanopure water and lyophilized for 48hours and stored at −20° C. until use. The concentration of PVA used toform the emulsion, as well as the sonication amplitude and duration canbe optimized to formulate particles having desired size and loading ofthe siRNA molecules. In an alternative approach, the siRNA payload inthe nanoparticle can be formulated as a spiegelmer as described by Vaterand Klussmann, Curr Opin Drug Discov Devel 6(2):253-61 (2003), which isincorporated herein by reference. This formulation delays theintracellular degradation of the RNA.

In another embodiment, the nanoparticle further comprises a magneticallyresponsive particle such as SPION. In this embodiment, the magneticparticle permits controlled movement or transport of the nanoparticle byapplication of a magnetic gradient to a desired location in the innerear. Furthermore, the addition of SPION to the nanoparticle compositionrenders the particles visible on a MRI scan thereby permittingconfirmation of nanoparticle localization to the appropriate tissue.These features and benefits are described in more detail in Wassel etal., Colloids and Surfaces A: Physiochem Eng. Aspects 292: 125-130(2007), which is incorporated herein by reference.

SPION can be incorporated into a PLGA nanoparticle complex using theabove described method of Woodrow et al. Specifically, SPIONs, in arange from about 5-10 mg/mL, can be dispersed into the PLGA/chloroformsolution along with the siRNA molecules as described above. It should beunderstood that SPION is the preferred magnetically responsive particle,however, the current invention should not be understood as being limitedthereto and other magnetic particles could be used that render thenanoparticle magnetically responsive and permit visualization by MRI.SPION can be incorporated into any of the nanoparticle/siRNA complexesdescribed herein.

In another embodiment, an agent is added to the nanoparticle that willinduce proliferation of the supporting cells of the inner ear.Regeneration of hair cells through the silencing of Hes1 results intransformation of supporting cells in the organ of Corti to sensory haircells. Consequently, this transformation may decrease the number ofsupporting cells which could result in a loss of integrity and functionin the organ of Corti. As such, the current embodiment includes firsttreating the cells of the inner ear with a molecule which will induceproliferation of the supporting cells. Thus, in addition to the siRNA,the nanoparticle complex, with or without SPION, may alternativelyinclude molecules in a therapeutically effective dose that wouldcontribute to the regenerative effect by increasing proliferation of thesupporting cells. For example, this proliferative event could be inducedby enhancing Skp2 activity, decreasing p27Kip1 activity, or downregulation of other inhibitors of cell cycle progression andproliferation that have not been discovered as yet.

In another aspect of this embodiment, thrombin could be used to induceproliferation of the supporting cells. Thrombin upregulates Skp2,cyclins D and A, and MiR-222, which effectively decreases activity ofp27Kip1. Therefore, in one embodiment, thrombin, either as a separatelyadministered protein or as a therapeutic payload is combined with thesiRNA payload in a nanoparticle. Thrombin is preferably administered24-48 hours prior to the administration of the siRNA. In the case wherethe thrombin and siRNA molecules are combined as a double payload in asingle nanoparticle, the thrombin and the siRNA are preferablyincorporated in such a way so as to cause release of the thrombin 24-48hours before the siRNA. The thrombin would be included in thenanoparticle complex at about 1-2% w/w.

Another molecule that can benefit the regenerative effect of silencingthe Hes1 gene is the micro RNA MiR-222. The MiR-222 inducesproliferation of the supporting cells by down regulating p27Kip1. Thus,an alternative embodiment includes the addition of a therapeuticallyeffective amount of MiR-222 into the nanoparticle complex. In order toreceive the full benefit of MiR-222, it is released from the particleprior to the release of the siRNA.

In yet another embodiment, the nanoparticle complex further comprises asurface peptide decoration for Coxsackie/Adenovirus receptor or otherpeptide that enhances transfection.

In another aspect of the current invention, a method for regeneratingsensory hair cells of the inner ear is provided. The method comprisesthe step of applying a solution to the inner ear wherein said solutioncomprises an active agent sufficient to decrease the expression oftarget genes selected from the group consisting of Hes1, Hes5, andMAPK1. In a preferred embodiment, the active agent comprises siRNAmolecules encapsulated into a nanoparticle. In fact, any of thenanoparticle/siRNA complexes described herein can be utilized in thismethod.

The solution can be any sterile solution compatible with the inner ear.The solution would ideally be isotonic with the perilymph of thelabyrinth. In a preferred embodiment, the solution is artificialperilymph which as described in Chen et al., J Control Release,110(1):1-19 (2006), which is incorporated herein by reference. Briefly,the artificial perilymph, for example, consists of: NaCl (120 mM); KCl(3.5 mM); CaCl2 (1.5 mM); glucose 5.5 (mM); and HEPES (20 mM). The pH ofthe artificial perilymph can be adjusted with NaOH to 7.5. Otherpossibilities would include 5% dextrose in sterile water, sterilephysiologic saline, or phosphate buffered physiologic saline.

Infusion volumes are preferably in the range of 1-100 μl infused slowlyover a period of 10 to 100 minutes. Furthermore, infusion could beextended using a microinfusion apparatus in the range of 1-20 μl perhour for days or weeks if needed. Multiple infusions could be repeatedif required.

In a preferred embodiment, the solution comprises nanoparticles carryingan siRNA payload suspended therein. The nanoparticle suspension can beprepared by mixing the required amount of nanoparticles in the solutionin 0.5 to 5 mg/mL concentration range. It can be sonicated for fewseconds to disperse the nanoparticles in the solution and stored around2-4° C. range to avoid any aggregation of nanoparticles.

The solution can be applied to the inner ear using a number of differentmethods. In one embodiment, the solution can be administered by directinjection through the round window membrane (RWM) or by infusion througha temporary or permanent cannula placed through the RWM. The infusion orinjection can be assisted through an attached microinfusion pump,dialysis apparatus, or fluid exchange system. Similar injection orinfusion technology could also be applied to the oval window, and/or theoval window ligament or annulus. The injections or infusion couldfurther be accomplished through a cochleostomy or other opening into theboney labyrinth such as one of the semicircular canals. Alternatively,the cortical bone could be removed over the labyrinth and a particlecontaining gel could be applied over the decorticated bone forintraosseous delivery. The particles could also be deliveredsystemically through intravenous or intrarterial administration.

In another embodiment, nanoparticle administration involves the use of amicro-catheter, such as the intra EAR round window catheter (RWMcCath™). In this method, such a catheter is introduced (such as via atympanotomy directly or endoscopically) into the middle ear from the earcanal and the distal tip of the catheter is placed immediately adjacentto the round window membrane. The nanoparticle solution is then passedinto the catheter and is brought into intimate association with theround window membrane facilitating diffusion of the nanoparticlesolution into the inner ear. These micro-catheters allow continualcontrolled pharmaceutical delivery to the round window membrane of themiddle ear and can remain in place for up to twenty-nine days (accordingto one micro-catheter use protocol).

In another embodiment, the nanoparticle solution can be applied to themiddle ear wherein the method further comprises the step of applying amagnetic force to enhance the transport of the particles across theround window membrane and into the inner ear. In this embodiment, thesiRNA loaded nanoparticle further comprise SPION or other magneticallyresponsive particles. The use of magnetic force to direct nanoparticlesinto the inner ear has been described in U.S. Pat. No. 7,723,311 andUnited States patent application publication nos. US 2004/0133099 and US2005/0271732, all of which are incorporated herein by reference.Preferably, the solution containing the nanoparticles would beadministered to the middle ear at the surface of the round windowmembrane (RWM) wherein a magnetic force is applied to drive thenanoparticles through the RWM and into the inner ear region.Alternatively, magnetic enhanced delivery can be applied to the ovalwindow (OW) niche and annular ligament.

More specifically, middle ear delivery of magnetically responsivenanoparticles is facilitated by, for example, a transtympanic injectionof the nanoparticle solution into the middle ear (such as via thetympanic membrane using a tympanotomy approach). To this end, a onecubic centimeter tuberculin syringe attached to a 27-gauge spinal needleis inserted into the tympanic membrane for intratympanic delivery ofnanoparticles. Delivery from the middle ear to the inner ear, across theround window membrane, is promoted by an externally placed magneticfield in the ear canal which drives the magnetically responsivenanoparticles across the round window membrane into the inner earfluids.

Alternatively, powerful permanent magnets could be placed on the surfaceof the boney labyrinth within the middle ear or mastoid cavity as amethod to magnetically capture the particles and concentrate them in thecirculation of the inner ear.

In an alternative embodiment, high frequency or low frequency soundexposure to the ear is used to further enhance the delivery of particleor drugs through the RWM or OW.

In yet another embodiment of the inventive method, the nanoparticlesolution is administered at the time of placement of a cochlear implantor any other intracochlear device or other occasion for opening thecochlea. The treatment could be made at the time of device placement totake advantage of the cochleostomy needed for device insertion.Alternatively the therapeutics described in this application could beinfused through a drug delivery catheter built into a device, as aseparately inserted device, or as a slow release polymer coating on thedevice that is inserted into the cochlea. In this example, a cochlearimplant electrode could be designed with a built in drug deliverycannula and an attached micropump as part of the implanted device.

In another embodiment, the nanoparticle bearing the appropriatetherapeutic could be injected at the time of insertion of the cochlearimplant with a temporary cannula, via a built-in cannula insertedthrough the cochleostomy or through the round window membrane.Alternatively, the cochlear implant electrode surface could be coatedwith a nanoparticle polymer containing the desired siRNA for slowrelease delivery by diffusion. The coating is preferably a polymercoating to protect and control the release of the siRNA. For example,PEG, PLGA or a combination of thereof may be used according to the drugrelease requirements. Regardless, the coating should be biocompatibleand be able to survive sterilization procedures and possess an extendedshelf life.

EXAMPLES

The composition and methods disclosed herein are proposed for thetreatment of both hearing and balance disorders. The Examples providedherein below provide scientific data to support the use of the disclosedcomposition and methods in replacement, protection and/or regenerationof sensory hair cells in the inner ear, and more specifically the haircells of the cochlear or vestibular labyrinth sensory epithelia. Thesenew hair cells have sufficiently normal anatomic orientation so as to befunctional to improve balance and hearing sense.

For all Examples below involving the use of siRNA molecules directed toHes1 mRNA (Hes1 siRNA) and Hes5 mRNA (Hes5 siRNA), a pool of three siRNAmolecules was utilized. For Examples siRNA molecules directed to MAPK1mRNA (MAPK1 siRNA), a single RNA molecule was utilized. The sequencesfor the siRNA molecules utilized in the following Examples are providedin Tables 1, 2 and 3. The Hes1 siRNA and Hes5 siRNA molecules wereobtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). The MAPK1siRNA molecules were synthesized using GenScript. The method describedabove with regards to formulation of the siRNA-PLGA nanoparticle(Woodrow et. al.) was used for all relevant Examples.

TABLE 1  Sequences for Hes1siRNA molecules Target sequence on Hes1 mRNAsiRNA Sense siRNA Antisense (SEQ ID NO: 1) Strand strand 239-257SEQ ID NO: 3 SEQ ID NO: 4 5′ CAGCUGAUAUA 3′ ttGUCGACUAUAU AUGGAGAAtt 3′UACCUCUU 5′ 371-389 SEQ ID NO: 5 SEQ ID NO: 6 5′ GAAGGGCAAGA 3′ttCUUCCCGUUCU AUAAAUGAtt 3′ UAUUUACU 5′ 1363-1381 SEQ ID NO: 7SEQ ID NO: 8 5′ GAUGCCAAAGA 3′ ttCUACGGUUUCU UGUUUGAAtt 3′ ACAAACUU 5′

TABLE 2  Sequences for Hes5 siRNA Target sequence on Hes5 mRNAsiRNA Sense siRNA Antisense (SEQ ID 2) Strand strand 164-182SEQ ID NO: 9 SEQ ID NO: 10 5′ GCAUCAACAGC 3′ ttCGUAGUUGUC AGCAUAGAtt 3′GUCGUAUCU 5′ 726-744 SEQ ID NO: 11 SEQ ID NO: 12 5′ GGUCAUUCUUA 3′ttCCAGUAAGAA GAGAAUGUtt 3′ UCUCUUACA 5′ 1141-1159 SEQ ID NO: 13SEQ ID NO: 14 5′ CGAUGAUCCUU 3′ ttGCUACUAGGA AAAGGAUUtt 3′ AUUUCCUAA 5′

TABLE 3  Sequences for MAPK1 siRNA Target sequence on MAPK1 mRNA siRNA Sense siRNA Antisense (SEQ ID NO: 17) Strand strand 1083-1101SEQ ID NO: 15 SEQ ID NO: 16 5′ UGCUGACUCCA 3′ CAGAGCUUUGGA AAGCUCUG 3′GUCAGCA 5′

Example 1

The objective of this study was to demonstrate that decreasing Hes1expression results in an increased number of hair cells and hair bundlesfollowing neomycin-induced cell death.

Utricular maculae from 300 gram guinea pigs were explanted, cultured invitro for 1 day, and exposed to neomycin for 48 hours and then treatedwith scrambled (control) or Hes1 siRNA (20 nmolar) and cultured foranother 5 days. Tissues were evaluated with confocal microscopy andtransmission electron microscopy (TEM).

FIG. 1 depicts TEM images taken from utricles of the scrambled RNAcontrol (A, E), neomycin treatment (B, F), and the neomycin plus Hes1siRNA treatment (C, D, G, H) groups. Images in the lower row are highermagnification of images from the upper row. Two layers of cells arefound in the utricle of the normal control group (A). The hair cells(type 1-T1, and type 2-T2, upper layer) demonstrate hair bundles (arrowsin A, E) and cuticular plates (stars in A, E). Supporting cells(arrowhead in A) contact the basement membrane. One hair cell is foundin the utricle of the neomycin treatment group, no hair bundle is shownat apex of this cell (HC in B). A continuous microfilament band hasformed at the apex of cells in this region (stars in B and F). Few hairbundles were seen (arrow in F). Two layers of cells are found in someregions of the utricular maculae of the neomycin plus Hes1 siRNAtreatment group (C). Hair cells at the upper layer have hair bundles(arrows in C and G) and cuticular plates (stars in C and G) whilesupporting cells are found in a lower layer (arrowhead in C). One layerof cells is also found in some regions of the utricular maculae of theneomycin plus siRNA treatment group (D and H). These cells have haircell bundles (arrows in H) and cuticular plates (star in H) and yetdirectly contact the basement membrane (D). Scale bars in A-H are 2microns.

The results provided in FIG. 1 below demonstrate that reducing theexpression of Hes1 following neomycin-induced hair cell death increasesthe number of hair cells and hair bundles in explanted guinea pigutricular maculae.

Example 2

The purpose of this study was to demonstrate the ability to deliver PLGAnanoparticles containing plasmid DNA-luciferase (pDNA) and SPION acrossthe round window membrane (RWM) of guinea pig in vivo using externalmagnetic forces.

Two microliters of solution containing PLGA nanoparticles carrying pDNAand SPION payload were placed on the RWM of one ear for each of the 12adult guinea pigs tested and 6 of the 12 guinea pigs were exposed toexternal magnetic forces estimated to be about 0.4 Tesla. The RWM nichesand cochlea of the subjects were then carefully isolated, separated andexposed to Protease K degradation for 12 hours at 50° C. in 0.1% SDS,Tris-EDTA (pH 8.0) buffer. Then pDNA was extracted from these tissues byphenol-chloroform denaturation of leftover proteins and precipitatedwith pre-chilled ethanol overnight. Total pDNA was resuspended to 100 μland 5 μl was used in each RT-PCR reaction. PCRs were set up usingInvitrogen Express One-Step SYBR Green PCR Master Mix in 20 μlreactions. Primers were designed using the Genscript software(Luciferase forward primer 5′-TGGAGAGCAACTGCATAAGG-3′ and reverse primer5′-CGTTTCATAGCTTCTGCCAA-3′). At the same time, a standard curve wasgenerated by running the same RT-PCR with templates of serial dilutionsof pDNA-luciferase (0.01 pg˜1 ng/ml). Melting curves and PCRs were runon an Eppendorf Realplex machine and results analyzed with Realplex DataProcessing software. The absolute pDNA amount in each sample wascalculated based on the standard curve. Each sample was run in tripletand Cycle Threshold values differing by a standard deviation greaterthan 0.5 were removed from the analysis, before being averaged tocalculate the pDNA amount.

FIG. 2 provides a bar graph that compares the control and magneticassisted transport. Results are expressed as means of six experiments(six control animals without magnet exposure and 6 animals with magnetexposure)±S.E.M (Standard Error of the Means). The statisticalcomparison was performed using paired, two-tailed Student's t-tests.

The results in FIG. 2 demonstrate that application of external magneticforce resulted in a significant 3.5-fold increase in perilymph/cochleardelivery of the pDNA (p<0.05). In addition, no pDNA was found in theears of animals opposite to the surgical ear where the pDNA was applied.This confirms the successful transport of a nucleic acid payload acrossthe RWM using this method.

Example 3

The purpose of this study was to confirm whether Hes1 siRNA delivered toexplanted guinea pig utricular tissue was effective to increase theproduction of new hair cells (immature-appearing hair cells) followingneomycin or 4-HNE induced injury.

Utricles were dissected from six week-old pigmented guinea pigs andexposed to the toxins neomycin (1 mM) or 4-HNE (200 μM) following thegeneral methodology described by Quint et al., Hear Res 118(1-2):157-67(1998), which is incorporated by reference herein. 48 hrs hours latercultures were placed in toxin-free medium, and transfected with eitherHes1 siRNA (24 pmoles/20 nM) or scrambled dsRNAs (24 pmoles) (control)using transfection agent jet SI (Polyplus-Transfection Inc., New York,N.Y.) for 24 hours. Tissues were then cultured another three days infresh medium without siRNA. Utricles were fixed in 4% paraformaldehydefor 1 hour at room temperature, washed 3 times with PBS, permeabilizedwith 0.05% Triton X-100 in PBS for 30 minutes, and then washed 3 timesin PBS. Explants were labeled with TRITC-conjugated phalloidin (3μg/mL), for 45 minutes in the dark at room temperature.Phalloidin-labeled hair cells (HCs) were observed using an Olympus BX-51epifluorescent microscope with a yellow excitation filter set at 560 to590 nm. Immature appearing hair cells (IAHCs) were counted from 6 to 102500 μm² fields for each explant (n=3-4 utricles per condition). IAHCshad very short uniform stereocilia bundles with or without a prominentkinocilium. IAHCs were quantified and expressed as a mean value+SEM(standard error mean). ANOVA with an LSD post hoc test was used tocompare the means between different conditions. A p value of <0.05 wasconsidered significant.

FIG. 3 provides quantitative results of the number of IAHCs intoxin-exposed-explants treated with Hes1 siRNA compared to toxin exposedexplants not treated with Hes1 siRNA and explanted utricles treated withscrambled siRNA (controls not exposed to toxin).

The data in FIG. 3 demonstrates that application of Hes1 siRNAsignificantly increases the number of new hair cells (IAHCs) inexplanted adult guinea pig utricular tissue that has been treated witheither neomycin or 4-HNE (p<0.001). The control data demonstrates thatunder normal conditions and following toxin injury, the cells only havelimited, natural capability to produce new hair cells. However,silencing Hes1 gene expression significantly increases the capability ofthe cells to produce new hair cells.

Example 4

The objective of this study was to determine whether it is possible tovisualize the PLGA/SPION nanoparticles non-invasively using 7 Tesla MRIscanning.

7 Tesla MRI images were taken from guinea pigs (a) not exposed to ananoparticle (control cochlea from the ear opposite to that which wasexposed to nanoparticle), (b) administered solution to the RWMcontaining PLGA/SPION nanoparticles in the absence of magnetic force and(c) administered solution to the RWM containing PLGA/SPION nanoparticlesin the presence of magnetic force. The cochlea were harvestedimmediately after a 45-minute exposure to PLGA-SPION nanoparticles andsubjected to 7 Tesla MRI scanning as described by Towner et al.,Molecular Imaging 6(1):18-29 (2007); Towner et al., Tissue Eng Part AAugust 7 (2009) [Epub ahead of print]; Towner et al., Tissue Eng Part A,January 10 (2010) [Epub ahead of print](hereinafter collectivelyreferred to as the “Towner references”), which are incorporated byreference herein. Although the images in FIGS. 4 and 5 were taken fromextracted cochlea, the Towner references in light of the results ofExample 4 adequately demonstrate the possibility of in vivo imagingusing the PLGA/SPION nanoparticle complex. T1 and T2 map values wererecorded and results are depicted in FIGS. 4 and 5.

As demonstrated in FIGS. 4 and 5, there was a decrease in T1 and T2values in the basal turn of the cochlea in animals exposed to thePLGA/SPION nanoparticles. Furthermore, this decrease in T1 and 12 wasfurthered by the presence of an external magnetic field. This indicatesthe presence of the nanoparticles in the cochlea and provides evidencethat such a detection system can be used to confirm tissue exposure tothe nanoparticle. Furthermore, this study demonstrates that applicationof an external magnetic force can be used to concentrate PLGA/SPIONnanoparticles in a particular region of the inner ear.

Example 5

The objective of this study was to demonstrate that the Hes1 siRNAmolecules used in the current studies are effective to decrease Hes1mRNA levels.

Cochlear tissues from P3 CD-1 mouse pups were dissected out and culturedin a 35 mm dish with collagen gel drops on the bottom. After initialincubation for 24 hours, the cochleae were transfected with HES1 siRNA(20 nM) using jetSI transfection reagents (Polyplus Inc.). The controltissues were treated with the same concentration of scrambled siRNA. Twodays later, all the tissues were placed into fresh medium. The cultureswere maintained for 7 days in vitro. Cochlear organotypic cultures werewashed in PBS and the total RNAs were extracted with TRIzol reagent(Invitrogen). Hes1 mRNA levels were analyzed by qRT-PCR on an Eppendorfrealplex PCR machine.

As depicted in FIG. 6, the Hes1 siRNA molecules used in this studyresulted in approximately a 75% decrease in Hes1 mRNA.

Example 6

The purpose of this study was to determine whether Hes1 siRNA iseffective in increasing the number of hair cells in the cochleafollowing injury with 4-HNE.

Organ of Corti were dissected from P3 CD-1 mouse pups and cultured oncollagen gel drops in 35 mm petri dishes in DMEM plusinsulin-transferrinselenite supplement (Sigma I-884). 4-hydroxynonenal(4-HNE) (200 μM) was added into the culture medium 24 hours later andthe control tissues were kept in drug free medium. 24 hours later, thetissues were transfected with Hes1 siRNA (20 nM in 1.2 ml) (24 pmol)using jetSI (Polyplus) transfection reagents at 2 mM and then added tothe DMEM medium. Fresh medium without siRNA or 4-HNE was applied to allthe tissues 48 hours later. After culturing for another 2 days, all thetissues were harvested and fixed with 4% paraformaldehyde andimmunostained with myosin Vila antibody and Phalloidin-TRITC (F-actinstaining). Hair cell counting and identification were carried out underfluorescence microscopy (Olympus BX 51 florescence microscope) with a40× objective lens. To be counted as a hair cell after toxin exposurethe cell had to evidence a cuticular plate, be myosin 7 positive, andbear a stereocilia bundle.

In the organ of Corti, transfection of Hest siRNA resulted in anincrease in hair cells in the Organ of Corti following injury with4-HNE. As depicted in FIG. 7, 4-HNE significantly decreased (p<0.05) thenumber of hair cells in the cochlea. Inhibition of Hes1 gene expressionthrough RNAi resulted in significant (p<0.05) regeneration of hair cellsfollowing injury. These results demonstrate that reducing the expressionof Hes1 is effective to increase the number of hair cells in the organof Corti under normal conditions as well as in response to injury with4-HNE.

Example 7

The purpose of this experiment is to demonstrate that PLGA nanoparticlesloaded with Hes1 siRNA is effective to increase the number of hair cellsfollowing injury.

Organs of Corti were dissected from P3 CD-1 mouse pups and cultured oncollagen gel drops in 35 mm petri dishes in DMEM plusinsulin-transferrinselenite supplement (Sigma I-884). The followingexperimental conditions were examined: (1) Control (n=6); (2) 4-HNE (200μM) (n=6); (3) PLGA nanoparticles (NPs) loaded with Hes1 siRNA (50μg/ml) (n=6); (4) 4-HNE (200 μM)+PLGA NPs loaded with Hes1 siRNA (1μg/ml) (n=6); (5) 4-HNE (200 μM)+PLGA NPs loaded with Hes1 siRNA (10μg/ml) (n=6); (6) 4-HNE (200 μM)+PLGA NPs loaded with Hes1 siRNA (50pig/ml) (n=6); (7) 4-HNE (200 μM)+PLGA NPs loaded with Hes1 siRNA (100μg/ml) (n=2); (8) 4-HNE (200 μM)+PLGA NPs loaded with control scrambledsiRNA (scRNA) (50 μg/ml) (n=6); (9) 4-HNE (200 μM)+PLGA NPs loaded withscRNA (100 μg/ml) (n=1). Cells were incubated for 24 hours in culturemedium with or without 4-HNE (200 μM) and then treated with PLGAnanoparticles loaded with Hes1 siRNA or scRNA. The culture medium wasreplaced every 48 hours such that the cells were exposed to three roundsof treatment with the siRNA containing-PLGA nanoparticles. All tissueswere harvested at day 8, fixed with 4% paraformaldehyde, andimmunostained with myosin Vila antibody and Phalloidin-TRITC (F-actinstaining). Inner and outers hair cells in the middle turn of OC werecounted and identified under fluorescence microscopy (Olympus BX 51florescence microscope) with a 40× objective lens. To be counted as ahair cell after toxin exposure, the cell had to demonstrate thefollowing characteristics: (1) a cuticular plate; (2) myosin 7 positive;(3) a stereocilia bundle.

FIG. 8 demonstrates the number of inner (IHC) and outer hair cells (OHC)in the middle turn of OC cultures of the various experimental groupsdescribed above. Data is expressed as mean±SD. 4-HNE exposuresignificantly decreased OHC numbers compared to normal controls.Treatment of non-ototoxin damaged OC cultures with the Hes1 siRNAnanoparticle (Hes1 NP) only (50 μg/ml) increased the hair cell numbercompared to normal control (p=0.02). Treatment of ototoxin-damaged (200μM 4-HNE) OC cultures with 50 or 100 μg/ml Hes1 NPs significantlyincreased OHC number compared to lower dose of Hes1 NPs (1 or 10 μg/ml,p=0.0001). As expected, scRNA nanoparticles (50 or 100 μg/ml) had noimpact on hair cell number in the OC exposed to 4-HNE.

In conclusion, treatment with PLGA nanoparticles encapsulated with Hes1siRNA increased the number of hair cells in the Organ of Corti.

Example 8

The purpose of this study was to determine whether siRNA directed toMAPK1 assisted by transfection agent jetSI (MAPK1 siRNA) or PLGAnanoparticles (MAPK1 siRNA-NPs) is effective in preventing hair celldeath in the saccules following injury with neomycin.

Saccules were dissected from P3 CD-1 mouse pups and cultured on collagengel drops in 35 mm petri dishes in DMEM plus insulin-transferrinselenitesupplement (Sigma I-884). 24 hours later, the culture medium wasreplaced with culture medium having a final concentration of 4 mMneomycin in the absence or presence of MAPK1 siRNA (standardtransfection reagent: 25 nM; 50 nM; 75 nM; and 100 nM) (PLGAnanoparticle:167 μg/ml; 333 μg/ml; 500 μg/ml; and 667 g/ml). Controlgroups were maintained in drug free medium. After culturing for a totalof 8 days, all the explants were harvested and fixed with 4%paraformaldehyde for 1 hour at room temperature, washed 3 times withPBS, permeabilized with 0.05% Triton X-100 in PBS for 30 minutes, andthen washed 3 times in PBS. Explants were labeled with TRITC-conjugatedphalloidin (3 μg/ml) for 45 minutes in the dark at room temperature.Phalloidin-labeled HCs were observed using an Olympus BX-51epifluorescent microscope with a yellow excitation filter set at 560 to590 nm. Hair cells (HCs) were counted from four 2500 μm² fields for eachexplant (n=3-5 saccules per condition). HCs were quantified andexpressed as a mean value±SEM (standard error mean). ANOVA with an LSDpost hoc test was used to compare the means between differentconditions. A p value of <0.05 was considered significant.

FIG. 9 depicts hair cell numbers in organotypic cultures of saccules innormal control, neomycin (4 mM), neomycin (4 mM)+MAPK1 siRNA, andneomycin (4 mM)+MAPK1 siRNA-NPs. Treatment with neomycin (4 mM)significantly decreased the number of hair cells in all groups ascompared to controls (p<0.01). However, treatment with MAPK1 siRNA (50nM or 75 nM, p<0.05) or MAPK1 siRNA-NPs (167 μg/ml, 333 μg/m, 500 μg/mlor 667 μg/ml) significantly increased (p<0.05) the number of survivinghair cells compared to treatment with neomycin (4 mM) alone. Increasingthe concentration of MAPK1 siRNA or MAPK1 siRNA-NPs did notsignificantly effect the number of hair cells (p>0.05). These resultsindicate that MAPK1 siRNA administered with either standard transfectionagent or encapsulated in PLGA nanoparticles successfully prevents haircell death caused by neomycin in organotypic cultures of saccules.

Although the invention has been described in connection with theembodiments disclosed herein, it should be understood that thisapplication is not intended to be limited to these embodiments and mayencompass other variations, uses, or adaptations of the inventionincluding such that are known or customary practice within the art whichare intended to be within the scope of the appended claims.

What is claimed is:
 1. A composition for regenerating hair cells of theinner ear comprising: (a) magnetically responsive nanoparticlescomprising a biodegradable polymer and an siRNA molecule that decreasesexpression of a Hes5 gene in a tissue of the inner ear and (b) apharmaceutically acceptable carrier.
 2. The composition of claim 1,wherein the biodegradable polymer is poly(lactic-co-glycolic acid). 3.The composition of claim 1, wherein the nanoparticles comprisesuperparamagnetic iron oxide.
 4. The composition of claim 1, wherein thenanoparticles comprise from about 500 to about 1000 siRNA molecules pernanoparticle.
 5. The composition of claim 1, wherein the nanoparticlesare dispersed in the pharmaceutically acceptable carrier.
 6. Thecomposition of claim 5, in which a concentration of the siRNA moleculesper milliliter of dispersion ranges from about 50 μg/mL to about 100μg/mL.
 7. The composition of claim 1, wherein the pharmaceuticallyacceptable carrier is selected from a group consisting of artificialperilymph, 5% dextrose in sterile water, sterile physiologic saline, andphosphate-buffered physiologic saline.
 8. A method of regenerating haircells of an inner ear of a mammal comprising: applying to the inner carof a mammal in need thereof an amount of biodegradable nanoparticlescomprising an siRNA molecule that decreases expression of a Hes5 gene ina tissue of the inner ear effective to regenerate hair cells in an innerear of said mammal.
 9. The method of claim 8, wherein the nanoparticlesare magnetically responsive.
 10. The method of claim 8, wherein theapplying comprises using magnetic force to transport the nanoparticlesacross the round window membrane.
 11. A composition for regeneratinghair cells of the inner ear comprising: (a) magnetically responsivenanoparticles comprising a biodegradable polymer and an siRNA moleculethat decreases expression of a MAPK1 gene in a tissue of the inner earand (b) a pharmaceutically acceptable carrier.
 12. The composition ofclaim 11, wherein the biodegradable polymer is poly(lactic-co-glycolicacid).
 13. The composition of claim 11, wherein the nanoparticlescomprise superparamagnetic iron oxide.
 14. The composition of claim 11,wherein the nanoparticles comprise from about 500 to about 1000 siRNAmolecules per nanoparticle.
 15. The composition of claim 11, wherein thenanoparticles are dispersed in the pharmaceutically acceptable carrier.16. The composition of claim 15, in which a concentration of the siRNAmolecules per milliliter of dispersion ranges from about 50 μg/mL toabout 100 μg/mL.
 17. The composition of claim 15, wherein thepharmaceutically acceptable carrier is selected from a group consistingof artificial perilymph, 5% dextrose in sterile water, sterilephysiologic saline, and phosphate-buffered physiologic saline.
 18. Amethod of regenerating hair cells of an inner ear of a mammalcomprising: applying to the inner ear of a mammal in need thereof anamount of biodegradable nanoparticles comprising an siRNA molecule thatdecreases expression of a Hes5 gene in a tissue of the inner eareffective to regenerate hair cells in an inner ear of said mammal. 19.The method of claim 18, wherein the nanoparticles are magneticallyresponsive.
 20. The method of claim 19, wherein the applying comprisesusing magnetic force to transport the nanoparticles across the roundwindow membrane.